Application of tetrahydropiperine in preparation of medicine for preventing / treating liver injury and liver fibrosis
By using tetrahydropiperine to inhibit the expression of TGF-β1 and block the CYP2E1 signaling pathway, the problem of activation of TGF-β1 signaling pathway in liver fibrosis was solved, and effective treatment of liver fibrosis was achieved.
Patent Information
- Application Number
- CN202411824583.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art is difficult to effectively inhibit the expression of transforming growth factor-β1 (TGF-β1) or block its signaling pathways, resulting in difficult control of the progression of liver fibrosis.
Tetrahydropiperine is used as a fibroblast inhibitor to exert therapeutic effects on liver fibrosis by inhibiting the expression of TGF-β1 and blocking the CYP2E1 signaling pathway.
Tetrahydropiperine significantly reduced the expression of α-SMA, P450, PTGS2, Caspase 3, and ICAM1 proteins in HSC-T6 cells, reduced the expression of ROS, and had good anti-hepatic injury and liver fibrosis activity.
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Figure CN119606964A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of medicines, and in particular to application of tetrahydropiperine in preparing medicines for preventing / treating liver damage and liver fibrosis. Background Art
[0002] Hepatic fibrosis (HF) is a common pathophysiological process in the development of chronic liver disease into end-stage liver diseases such as cirrhosis, liver cancer, and liver failure. It is characterized by the progressive accumulation of extracellular matrix (ECM) in the liver and is reversible. The level of liver fibrosis is related to liver function and is a major risk factor for hepatocellular carcinoma (HCC). Studies have found that the occurrence and development of liver fibrosis is mainly caused by the activation and proliferation of hepatic stellate cells (HSCs). During HF, hepatic stellate cells are activated, producing a large amount of ECM components such as collagen type I (Col-I) and expressing high levels of alpha smooth muscle actin (α-SMA).
[0003] Transforming growth factor-β1 (TGF-β1) is one of the most critical cytokines in the process of liver fibrosis. It mainly activates Smad2 and Smad3 in cells by binding to receptors on the cell membrane, activating HSCs, promoting the diffuse proliferation and deposition of ECM deposition, and eventually developing into liver fibrosis. TGF-β1 promotes the expression of α-SMA in cells and is a key factor in activating HSCs. It plays an important role in the process of liver fibrosis. Therefore, how to design a new treatment plan to effectively inhibit the expression of TGF-β1 or block its signaling pathway is the key to preventing and treating liver fibrosis. Summary of the invention
[0004] The object of the present invention is to provide an application of tetrahydropiperine in the preparation of a medicament for preventing / treating liver damage and liver fibrosis, so as to solve the above-mentioned problems in the background technology.
[0005] To achieve the above object, the present invention provides the following technical solutions:
[0006] One of the technical solutions of the present invention is to provide an application of tetrahydropiperine in the preparation of a medicine for treating liver damage and liver fibrosis.
[0007] The second technical solution of the present invention is to provide an application of tetrahydropiperine in the preparation of a drug for preventing liver damage and liver fibrosis.
[0008] Preferably, the drug is a fibroblast inhibitor, which exerts its efficacy by inhibiting the proliferation activity of hepatic stellate cells.
[0009] The third technical solution of the present invention is to provide a fibroblast inhibitor containing tetrahydropiperine.
[0010] Preferably, the fibroblast inhibitor exerts its drug effect by inhibiting the expression of TGF-β1.
[0011] Preferably, the fibroblast inhibitor exerts its drug effect by blocking the CYP2E1 signaling pathway.
[0012] Preferably, tetrahydropiperine is the main active ingredient in the fibroblast inhibitor.
[0013] Preferably, the fibroblast inhibitor further comprises pharmaceutically acceptable excipients.
[0014] Preferably, the fibroblast inhibitor is in the form of tablets, pills, capsules, granules, powders or liquid preparations.
[0015] Piper longum is a perennial herbaceous climbing vine of the genus Piper in the family Piperaceae. It has antibacterial, anti-inflammatory, antiviral and metabolic regulating effects. Through modern pharmacological analysis, the active ingredient tetrahydropiperine was screened out from Piper longum as the research object, and P450, PTGS2, Caspase 3, and ICAM1 were selected from the key targets of Piper longum and liver fibrosis as the research object. It was found that tetrahydropiperine can play a protective role against liver damage by downregulating the CYP2E1 signaling pathway, which provides a certain theoretical basis for the treatment of liver fibrosis.
[0016] The beneficial technical effects of the present invention are as follows:
[0017] The present invention has found that the effective ingredient in the longum longum extract, tetrahydropiperine, can reduce the expression of α-SMA, P450, PTGS2, Caspase 3, and ICAM1 proteins in HSC-T6 cells by down-regulating the CYP2E1 signaling pathway, and play a mechanism of protecting liver damage, and has good anti-liver damage or liver fibrosis activity. When the tetrahydropiperine drug is treated, the expression of ROS is significantly reduced. Studies have found that the liver protection effect of tetrahydropiperine is stronger than that of piperine, and the protective effect on the liver gradually increases with the increase of drug concentration.
[0018] The experiment found that after the HSC-T6 cells were treated with tetrahydropiperine, the expression of α-SMA, P450, PTGS2, Caspase 3, and ICAM1 proteins could be lower than that of the piperine group. By detecting the cell activity with CCK-8, it was found that the cell activity was significantly reduced after the tetrahydropiperine drug treatment, and ROS detection found that the expression in the tetrahydropiperine group was significantly reduced. The present invention provides a certain theoretical basis for the prevention and treatment of liver fibrosis, and provides a new idea and method for the study of liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0020] Figure 1 This is the effect of the control group, piperine group and tetrahydropiperine group on the viability of HSCs cells.
[0021] Figure 2 This is a comparison of the effects of piperine and tetrahydropiperine on HSCs cell viability.
[0022] Figure 3 This is the intracellular ROS content of HSCs in the control group, model group, piperine group, and tetrahydropiperine group.
[0023] Figure 4 The figure shows the protein immunoblotting results of the control group, model group, and tetrahydropiperine group.
[0024] Figure 5 The protein expression levels of α-SMA (A), P450 (B), PTGS2 (C), Caspase 3 (D), and ICAM1 (E) in HSCs cells of the control group, model group, and tetrahydropiperine group.
[0025] Figure 6 The graphs show the protein immunoblotting results of the control group, model group, piperine group, and tetrahydropiperine group.
[0026] Figure 7 The protein expression levels of α-SMA (A), P450 (B), PTGS2 (C), Caspase 3 (D), and ICAM1 (E) in HSCs cells of the control group, model group, piperine group, and tetrahydropiperine group.
[0027] Figure 8 Macroscopic images of the livers of groups A, B, and C after treatment. DETAILED DESCRIPTION
[0028] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.
[0029] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.
[0031] The words “include,” “including,” “have,” “contain,” etc. used in the present invention are open-ended terms, meaning including but not limited to.
[0032] The study found that by adding TGF-β1 during cell culture, the expression of α-SMA in HSC-T6 was significantly increased, and the expression of P450, PTGS2, Caspase 3, and ICAM1 also increased significantly. By inhibiting the expression of TGF-β1, its signaling pathway for activating HSCs can be blocked, thereby reducing the synthesis and deposition of ECM, which helps to reduce the degree of liver fibrosis and delay the progression of the disease. Due to the important role of TGF-β1 in liver fibrosis, it has become a potential target for the treatment of liver fibrosis. By inhibiting the expression of TGF-β1 or blocking its signaling pathway, new strategies and methods can be provided for the treatment of liver fibrosis.
[0033] The "room temperature" in the present invention is 10-30°C unless otherwise specified.
[0034] All raw materials used in the following examples and comparative examples of the present invention are commercially available products.
[0035] The chemical formula of tetrahydropiperine is C 17 H 23 NO 3 The tetrahydropiperine used in the present invention is purchased from Shanghai Yuanye Company, and the acquisition method is not limited to the above method.
[0036] Example 1
[0037] In vitro and in vivo experiments were used to verify the protective effect of tetrahydropiperine on liver injury / liver fibrosis:
[0038] 1. Experimental methods:
[0039] 1. Cell Culture and Grouping
[0040] Rat hepatic stellate cells (HSC-T6) were used for the study. HSC-T6 cells in the logarithmic growth phase were collected to prepare single-cell suspensions (the culture medium was prepared with 10% FBS, 1% double antibody and 89% DMEM culture medium). 5×10 3 Cells were inoculated into 96-well culture plates at a density of 1 cell / mL and incubated at 37°C and 5 vol% CO 2 Culture at a constant temperature in an environment. When the cell density grows to 50-60%, discard the culture medium and replace it with fresh culture medium to obtain the cultured cell sample, then add drugs to the culture wells (5 replicate wells per group), culture in different groups of culture methods, and then place in a cell culture incubator to continue incubation for 24 hours to obtain the sample to be tested. Among them, the culture methods of different groups are as follows:
[0041] Control group: HSC-T6 cells were not treated and recorded as Control.
[0042] Model group: HSC-T6 cells were treated with TGF-β1 (5 ng / mL) and recorded as Model.
[0043] Piperine group: After HSC-T6 cells were treated with TGF-β1 (5 ng / mL) for 24 h, piperine was added to the concentrations of 10, 20, and 40 μmol / L, respectively, and HSC-T6 cells were treated for 24 h. The treatment groups with different concentrations were recorded as Piperine-L, Piperine-M, and Piperine-H, respectively.
[0044] Tetrahydropiperine group: After HSC-T6 cells were treated with TGF-β1 (5 ng / mL) for 24 h, tetrahydropiperine was added to the concentrations of 10, 20, and 40 μmol / L, respectively, and the HSC-T6 cells were treated for 24 h. The treatment groups with different concentrations were recorded as THP-L, THP-M, and THP-H, respectively.
[0045] 2. CCK-8 detection of HSC-T6 cell proliferation activity
[0046] Add 10 μL of CCK-8 solution to each well of the sample to be tested (the amount of CCK-8 solution added is 10% of the total liquid volume of each well), continue to culture for 2 hours, then place it in an ELISA reader at a wavelength of 450nm to detect the absorbance (A) value, and calculate the cell survival rate (%) according to the instructions and the measured absorbance. The test results are as follows Figure 1-2 shown.
[0047] 3. Cytoplasmic ROS Detection
[0048] Take the sample to be tested, remove the cell culture medium, wash twice with PBS, and add trypsin digestion solution to digest the cells. After digestion, add 1mL of serum-free medium, blow to make the cells fall off the 96-well plate, make a cell suspension and transfer it to a 2mL centrifuge tube. Add 10μM DCFH-DA fluorescent probe to each tube (except the control group), and then incubate in a 37℃ water bath in the dark for 30min, and mix several times to prevent the cells from sinking to the bottom. Then wash the cells 3 times with PBS to completely remove the DCFH-DA dye that has not entered the cells and remains in the solution. Finally, collect the cells of each group and use a multifunctional microplate reader for on-machine detection at an excitation wavelength of 488nm and an emission wavelength of 525nm.
[0049] 4. Western Blot
[0050] Protein immunoblotting is a new immunobiochemical technique developed on the basis of gel electrophoresis and solid phase immunoassay technology, which uses the specific binding of antigen and antibody to detect proteins in complex samples.
[0051] Take the sample to be tested, and obtain the protein sample solution after the protein extraction step. The protein sample solution and the pre-stained protein marker are loaded separately, and the standard is added to the first well to separate the protein by electrophoresis; the protein is transferred to the PVDF membrane, and the filter paper gel cellulose sandwich is assembled according to the instructions of the Bio-Rad protein transfer device, and the transfer is carried out at 4°C overnight under 30mA constant current conditions. The membrane is blocked and the antibody is incubated in 5% skim milk powder solution at room temperature for 1h to block the non-specific binding on the membrane; the blocked membrane is incubated with the primary antibody at room temperature for 1.5h for antigen-antibody binding; the HRP-labeled secondary antibody is added to bind the primary antibody, and the membrane is incubated at room temperature for 1h; the HRP-labeled GAPDH antibody is added to simultaneously detect the GAPDH content; the chemiluminescence method is used for detection, the membrane is incubated with the chemiluminescent substrate, and the specific strips of the obtained picture are analyzed after exposure and development; the gray value of the target protein is divided by the gray value of the internal reference GAPDH / Actin to correct the error, and the result represents the relative content of the target protein in the sample.
[0052] 5. In vivo animal experiments
[0053] The rats were raised for 8 weeks and then anesthetized and photographed;
[0054] SD rats were divided into group A, group B and group C. Group A was gavaged with 50 wt% CCl 4 The rats in group B were gavaged with a mixture of 20 mg / kg piperine and DMSO (the ratio of the added mixture to the rat body weight was 0.3 mL / 100 g), the rats in group B were gavaged with a mixture of 20 mg / kg piperine and DMSO (the ratio of the added mixture to the rat body weight was 0.1 mL / 100 g), and the rats in group C were gavaged with a mixture of 20 mg / kg tetrahydropiperine and DMSO (the ratio of the added mixture to the rat body weight was 0.1 mL / 100 g); then the SD rats were fed for 8 weeks, then anesthetized and photographed.
[0055] 2. Experimental results:
[0056] 1. CCK-8 detection of HSC-T6 cell proliferation activity
[0057] Figure 1 This is the effect of the control group, piperine group and tetrahydropiperine group on the viability of HSCs cells.
[0058] Figure 1 In the middle, *P<0.05, **P<0.01, ***P<0.001vs Control.
[0059] Figure 2 This is a comparison of the effects of piperine and tetrahydropiperine on HSCs cell viability.
[0060] Figure 2 In the middle, *P<0.05, **P<0.01, ***P<0.001Piperine vs THP.
[0061] Depend on Figure 1 It can be seen that compared with the HSC-T6 cell viability of the control group, the proliferation activity of HSC-T6 cells in the tetrahydropiperine group and the piperine group (10, 20, 40 μmol / L) was reduced in a dose-dependent manner, and the difference was statistically significant (P<0.05). Figure 2 It can be seen that compared with the piperine group, the proliferation activity of HSC-T6 cells in the tetrahydropiperine groups (10, 20, 40 μmol / L) was reduced (P<0.001).
[0062] 2. Expression of cytoplasmic ROS
[0063] Figure 3 This is the intracellular ROS content of HSCs in the control group, model group, piperine group, and tetrahydropiperine group.
[0064] Figure 3In, ###P<0.001Model vs Control; ***P<0.001**P<0.01, Piperine vs THP.
[0065] Depend on Figure 3 Comparison between the control group and the model group showed that after treatment with TGF-β1 (5 ng / mL), the expression of ROS in the cells was significantly increased; after treatment with tetrahydropiperine and piperine, the expression of ROS in HSC-T6 cells was significantly decreased, and the difference was statistically significant (P<0.05); compared with the HSC-T6 cells in the piperine group, tetrahydropiperine had a statistically significant effect on the decrease of ROS expression in HSC-T6 cells.
[0066] 3. Changes in the expression levels of α-SMA, P450, PTGS2, Caspase 3, and ICAM1 proteins in HSC-T6.
[0067] Figure 4 The figure shows the protein immunoblotting results of the control group, model group, and tetrahydropiperine group.
[0068] Figure 5 The protein expression levels of α-SMA (A), P450 (B), PTGS2 (C), Caspase 3 (D), and ICAM1 (E) in HSCs cells of the control group, model group, and tetrahydropiperine group.
[0069] Figure 5 Medium, #P<0.05, ##P<0.01, ###P<0.001vs Control, *P<0.05, **P<0.01, ***P<0.001vs Model.
[0070] Figure 5 In the figure, low dose, middle dose, and high dose represent the groups of HSC-T6 cells treated with piperine at concentrations of 10, 20, and 40 μmol / L, respectively.
[0071] Figure 4-5 The results showed that compared with the control group, the expression levels of α-SMA, P450, PTGS2, Caspase 3 and ICAM1 proteins in the model group were significantly increased (P<0.01); compared with the model group, the expression levels of α-SMA, P450, PTGS2, Caspase 3 and ICAM1 proteins in the tetrahydropiperine group were significantly decreased (P<0.05), among which the expression levels of α-SMA, P450, PTGS2, Caspase 3 and ICAM1 proteins in the high dose of tetrahydropiperine group were significantly decreased (P<0.01).
[0072] Figure 6 The graphs show the protein immunoblotting results of the control group, model group, piperine group, and tetrahydropiperine group.
[0073] Figure 7 The protein expression levels of α-SMA (A), P450 (B), PTGS2 (C), Caspase 3 (D), and ICAM1 (E) in HSCs cells of the control group, model group, piperine group, and tetrahydropiperine group.
[0074] Figure 7 In, ###P<0.001 vs Control; ***P<0.001Piperine vs THP.
[0075] Figure 6-7 The results showed that compared with the piperine group, the expression levels of α-SMA, P450, PTGS2, Caspase 3, and ICAM1 proteins in the tetrahydropiperine group were significantly decreased (P<0.001).
[0076] 4. In vivo animal experiment results
[0077] Figure 8 Macroscopic images of the livers of groups A, B, and C after treatment.
[0078] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. An application of tetrahydropiperine in the preparation of a medicine for treating liver damage and liver fibrosis.
2. An application of tetrahydropiperine in the preparation of a drug for preventing liver damage and liver fibrosis.
3. The use according to claim 1 or 2, characterized in that: The drug is a fibroblast inhibitor, and exerts its efficacy by inhibiting the proliferation activity of hepatic stellate cells.
4. A fibroblast inhibitor, characterized in that The fibroblast inhibitor contains tetrahydropiperine.
5. The fibroblast inhibitor according to claim 4, characterized in that The fibroblast inhibitor exerts its drug effect by inhibiting the expression of TGF-β1.
6. The fibroblast inhibitor according to claim 4, characterized in that The fibroblast inhibitor exerts its drug effect by blocking the CYP2E1 signaling pathway.
7. The fibroblast inhibitor according to claim 4, characterized in that Tetrahydropiperine is the main active ingredient in the fibroblast inhibitor.
8. The fibroblast inhibitor according to claim 4, characterized in that The fibroblast inhibitor further comprises pharmaceutically acceptable excipients.
9. The fibroblast inhibitor according to claim 4, characterized in that The dosage form of the fibroblast inhibitor is tablets, pills, capsules, granules, powders or liquid preparations.